Nucleotide analogues are useful tools for the investigation of interactions between DNA-binding proteins and DNA at a molecular level. Herein we describe the synthesis of the DNA-lesion analogue 2, which is required to determine the extent to which specific phosphodiesters in the DNA backbone contribute to the recognition of cyclobutane pyrimidine dimer DNA lesion by the dimer-specific repair enzymes DNA photolyases or T4-endonuclease V. The analogue 2 is a close structural mimic of cyclobutane pyrimidine dimers like 1, which are the major lesions induced upon irradiation of cells with UV light. Instead of the negatively charged phosphate link in 1, analogue 2 contains an uncharged but isosteric formacetal moiety. The analysis of this and other phosphodiester contacts is hoped to provide insight into the lesion recognition process, which is currently believed to require the flipping of the lesioned base out of the DNA double helix. The lesion analogue 2 is synthetically available in large quantities, which allowed us to establish a new, fast and sensitive DNA photolyase assay. A precise X-ray crystal structure analysis of the DNA-lesion analogue 2 is also presented. The structure underlines the isosteric character of 2 and reveals, in combination with the only other available X-ray crystal structure determined from a thymine-dimer triester analogue, interesting structural features of cyclobutane pyrimidine dimer lesions. We describe the incorporation of the lesion analogue 2 into oligonucleotides by using standard phosphoramidite chemistry. Initial enzymatic repair studies are reported with three different types of DNA photolyases. These studies show that the lesion analogue 2 is rapidly repaired by photolyases from Anacystis nidulans, Neurospora crassa and from the marsupial Potorous tridactylis. The enzymatic investigations indicate that all photolyases, including enzymes from higher organisms (P. tridactylis) accept the formacetal dimer as a lesion substrate and therefore could possess a similar DNA-lesion recognition process, in which the interaction with the central phosphate unit is only of limited importance.

have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 Â His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity.Keywords: Arabidopsis thaliana; class II DNA photolyase; DNA repair; enzymatic properties; spectroscopic properties.UV-B light causes two major types of DNA-damage, the formation of cyclobutane pyrimidine dimers (CPD) and the (6-4)-photoproduct [1±4]. Both types of damage can be directly reversed in a process called photoreactivation [5]. Photoreactivation is mediated by enzymes which use light energy in the UV-A/blue region for repair [2]. The first photolyases to have been studied and cloned are CPD-photolyases from microbial organisms (class I) [2]. All of them contain reduced FAD, which is necessary for catalytic activity. Besides FAD, class I photolyases contain a second chromophore, which is speciesdependent either: 5,10-methenyltetrahydrofolate (MTHF) or 8-hydroxy-5-deaza-riboflavin (8-HDF) [6±8]. The second chromophore determines the absorption characteristics of the enzyme. MTHF-containing photolyases have an absorption maximum around 384 nm, 8-HDF-containing photolyases have an absorption maximum around 438 nm [2]. Light energy absorbed by the second chromophore is transferred to the reduced FAD with high efficiency. The excited FADH 2 then transfers an electron to the CPD leading to a CPD radical anion, which is unstable. The carbon bonds of the cyclobutane ring are split and the electron is channelled back to the neutral flavin radical to regenerate FADH [23,24]. Sequence comparison between class I and class II CPDphotolyases revealed that class I and class II photolyases form separate and only distantly related groups of enzymes which have diverged early in evolution [25]. In contrast with class I CPD photolyases, little is known about the reaction mechanism of class II photolyases. In this paper we describe the overexpression of Arabidopsis CPD-photolyase in E. coli, its purification and analysis for chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite of this the purified enzyme has photoreactivating activity. E X P E R I M E N T A L P R O C E D U R E SMaterials FAD and riboflavin were from Sigma; d-phenylglycine was from Fluka. MTHF was synthesized according to the method of Rabinowitz [26]. Coenzyme F 420 purified from M. thermoautotrophicum was a gift of R. Hedderich (MPI fu Èr terrestrische Mikrobiologie, Marburg, Germany). Restriction enzymes, Taq polymerase and Klenow polymerase were from MBI Fermentas Correspondence to A.

Cyclobutane uridine and thymidine dimers with cis-syn-structure are DNA lesions, which are efficiently repaired in many species by DNA photolyases. The essential step of the repair reaction is a light driven electron transfer from a reduced FAD cofactor (FADH ) to the dimer lesion, which splits spontaneously into the monomers. Repair studies with UV-light damaged DNA revealed significant rate differences for the various dimer lesions. In particular the effect of the almost eclipsed positioned methyl groups at the thymidine cyclobutane dimer moiety on the splitting rates is unknown. In order to investigate the cleavage vulnerability of thymine and uracil cyclobutane photodimers outside the protein environment, two model compounds, containing a thymine or a uracil dimer and a covalently connected flavin, were prepared and comparatively investigated. Cleavage investigations under internal competition conditions revealed, in contrast to all previous findings, faster repair of the sterically less encumbered uracil dimer. Stereoelectronic effects are offered as a possible explanation. Ab initio calculations and X-ray crystal structure data reveal a different cyclobutane ring pucker of the uracil dimer, which leads to a better overlap of the pi*-C(4)-O(4)-orbital with the sigma*-C(5)-C(5')-orbital. Enzymatic studies with a DNA photolyase (A. nidulans) and oligonucleotides, which contain either a uridine or a thymidine dimer analogue, showed comparable repair efficiencies for both dimer lesions. Under internal competition conditions significantly faster repair of uridine dimers is observed.

DNA and RNA are the molecules which store the genetic information in every organism. Both macromolecules are severely damaged by a variety of exogenous and endogenous events. This leads to a loss of genetic information. Main lesions are strand breaks, apurinic and apyrimidinic sites and a variety of DNA bases with an altered structure. The result of these DNA modifications are cell death, mutations and in the worst scenario carcinogenous growth. In order to study the effect of DNA lesions on the structure of the DNA double helix, a variety of lesion building blocks were recently synthesized and incorporated into oligonucleotides. In addition, oligonucleotides which contain DNA lesions at specific sites are the basis for a detailed investigation of repair mechanisms that were developed by organisms in order to counteract the lethal effect of DNA damage. This review article describes the recent synthetic progress that has enabled the preparation of DNA lesion phosphoramidite building blocks. The synthetic procedures employed for their preparation and the methods used to incorporate these building blocks into oligonucleotides are described. The biological effect of each particular lesion is briefly recapitulated.

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